The application of antibiotics to the treatment of bacterial infections revolutionized modern medical practice. In the decades since, a combination of improperly controlled use and the remarkable ability of bacterial populations to develop resistance to these drugs has severely restricted the clinical usefulness of many antibiotics. We are now at a critical point where the majority of these drugs have known and sometimes extensive resistance, and few novel replacements or strategies to combat the resistance problem exist. The aminoglycoside antibiotics are one important example of a large group of drugs with wide application in clinical practice but where the problem of resistance continues to increase. Of great concern is the identification over the last several years of pathogenic bacteria with broad-spectrum high-level resistance conferred by enzymes that modify the aminoglycoside binding site on the RNA of the small ribosomal subunit. Two distinct families of aminoglycoside-resistance methyltransferases have been identified that modify 16S ribosomal RNA on nucleotide G1405 or A1408. Functionally analogous self-protection resistance enzymes of both types were earlier identified in aminoglycoside-producing bacteria, and it is thought that these genes have laterally transferred to the pathogenic bacteria. However, in both cases the resistance enzymes involved are very poorly structurally and biochemically characterized. This proposal describes experiments grouped into two connected aims that will directly address this deficiency. In Aim 1, we will determine the high-resolution X-ray crystal structures of members of both families of aminoglycoside-resistance methyltransferases (i.e. those targeting G1405 and A1408) from both aminoglycoside-producers and pathogenic bacteria. We will also characterize newly identified potential members of this resistance methyltransferase family that may provide further detailed insights into the origins of these enzymes in pathogenic bacterial populations. Finally, for each enzyme family, we will characterize and compare the binding of the essential methyl group donor molecule S-adenosyl-L-methionine (SAM) using mutagenesis and isothermal titration calorimetry (ITC). In Aim 2, we will fully dissect the mechanism of methyltransferase- small ribosome subunit recognition, which represents the best target for future specific enzyme inhibitors. We will determine critical amino acids within each enzyme for target specificity using site-directed mutagenesis in combination with binding and functional assays, map the docking site on the small ribosome subunit using structure probing and cross-linking experiments, and provide the first high-resolution view of an antibiotic resistance methyltransferase enzyme bound to the small ribosome subunit using X-ray crystallography. Together these studies will provide novel and fundamentally important insights into the structure and function of aminoglycoside-resistance methyltransferases and will lay a secure foundation for any future development of new strategies to counter the resistance they confer.